Protein synthesis occurs with a high degree of precision that depends on numerous components, such as ribosomal proteins, and standard and novel translation factors. The study of omnipotent suppression in the yeast Saccharomyces cerevisiae is one of the most fruitful approaches for identifying factors involved in translational ambiguity and for uncovering new components of the translational machinery of eukaryotic cells. Furthermore, the yeast S. cerevisiae represents an especially convenient model organism for investigating the translational apparatus of eukaryotes because the genes identified in yeast are usually evolutionarily conserved and have their counterparts in other eukaryotic organisms. Also, the powerful genetic and DNA recombinant methods available for yeast allows convenient disruption and modification of genes. The main aim of the proposed research project is to investigate the functions of proteins encoded by omnipotent suppressors, with an emphasis on SUP45 and SUP35, and to define their roles in the translational process. the Sup45 and Sup35 proteins will be purified and used in yeast cell-free translational systems and for studies of their interactions with aminoacyl- tRNA, GTP and mRNA. Mutations, deletions and oligonucleotide-directed mutagenesis will be used to study the structure-function relationship of these proteins. The specific role in the translational process and their interaction with other components of the translational apparatus will be investigated with cell-free systems. Studies of proteins interacting with the Sup45 and Sup35 proteins will be used as an attempt to define the interrelationships of translation with other processes in yeast. Because the SUP35 and SUP45 genes appear to affect different cellular processes including, respiration and cell cycle control, we plan to study the nuclear localization of the Sup35 and Sup45 proteins by cellular fractionation and by the method of indirect immunofluorescence, and to study of possible interaction of these proteins with DNA. The wild-type genes corresponding to the newly isolated SUP42 and SUP43, and possibly other omnipotent suppressors, will be sequenced. If they appear to be novel translational factors, their function will be further investigated by the approaches described above. Plasmid-mediated amplification of the wild-type genes, leading to omnipotent suppression, will be used as an approach for identification of novel components of translational apparatus involved in maintaining the translational accuracy in eukaryotes.